MEMBRANE SUBSTRUCTURE AND CELL DIVISION IN E. COLI

所有细胞都表现出生化反应，这种反应只在 它们的膜中有特定的位置。最基本的 问题是，为什么这些反应发生在一个部位，而不是 其他地方看似相同的位置。两个最重要的 其中在大肠杆菌中的这些过程是相关的 到细胞分裂：隔膜分裂平面的形成，以及 子代细胞的染色体分离。长距离的 目的是了解人类免疫缺陷的分子机制。 细胞控制着这些事件的位置特异性。这项建议 被设计用来确定大肠杆菌的膜是如何 这一机制。 将对以前证明的方法进行改进 膜泡可以分成更广泛的类别 而不是经典的内/外膜定义。这些方法 包括带电分离(通过 稀释琼脂糖)和大小(通过Sephacryl- 太棒了。还将开发其他技术，包括快速 基于凝集素亲和力的分析和分级。 这些方法将有助于回答以下问题：Do 蛋白质具有特定的序列，这些序列将它们引导到不同的 囊泡亚群；主要细胞分裂蛋白是否相互作用 在“分离亚区”中相互连接；而染色体 与特定的膜结构相互作用？第一个问题 将通过测量融合蛋白的位置来解决 报告酶碱性磷酸酶。剩下的问题 将通过：免疫印迹定位蛋白质在 野生型和突变型细菌的囊泡.定位 青霉素结合蛋白.使用 X174裂解蛋白E；核酸杂交检测DNA- 囊泡联合，使用ORIC和mini-F质粒作为 实验探头。 从长远来看，了解大肠杆菌是如何产生和维持的 其膜内的组织以及该结构如何贡献 可能成为理解细胞分裂的基础 生化过程在空间上是受限的，这是如何 排列调节细胞的生长和分裂，以及这些 可能会抑制这一过程，以控制细菌感染。

All cells exhibit biochemical reactions which occur exclusively at specific locations within their membranes. The fundamental question is why the reactions occur at one site instead of a seemingly equivalent location elsewhere. The two most significant of these processes in the bacterium Escherichia coli are related to cell division: formation of the septal division plane, and chromosomal segregation to daughter cells. The long-range objective is to understand the molecular mechanism by which the cell controls the site-specificity of these events. This proposal is designed to determine how the membrane of E. coli contributes to this mechanism. Improvements will be made in methods which previously demonstrated that membrane vesicles can be separated into classes more extensive than the classical inner/outer membrane definition. These methods include separation by charge (electrophoresis of vesicles through dilute agarose) and by size (chromatography through Sephacryl- SlOOO. Additional techniques will be developed, including rapid assays and fractionation based on lectin affinity. These methods will help answer the following questions: do proteins have specific sequences which direct them to different vesicle sub-populations; do major cell division proteins interact with one another in "septation subdomains;" and does the chromosome interact with a particular membrane structure? The first question will be addressed by surveying the location of proteins fused to the reporter enzyme, alkaline phosphatase. The remaining questions will be addressed by: immunoblot localization of proteins in vesicles of wild-type and mutant bacteria; localization of penicillin-binding proteins; genetic characterization using the X174 lysis protein E; and nucleic acid hybridization to detect DNA- vesicle associations, using oriC and mini-F plasmids as experimental probes. In the long term, knowing how E. coli creates and maintains organization within its membrane and how that structure contributes to cell division may form the basis for understanding how biochemical processes are spatially restricted, how this arrangement regulates cell growth and division, and how these processes may be inhibited to control bacterial infections.